162 ◾ Fundamental Food Microbiology
suspending medium contains cryoprotective agents to reduce cell death and injury during freezing
and thawing. The cell suspensions in metal containers are frozen, either by using dry ice–acetone
(–78°C) or liquid nitrogen (–196°C), and stored at that temperature. They are transported to processors, preferably by air, in Styrofoam boxes with sufficient amounts of dry ice. A processor stores
the containers at –20°C or below and uses it within the time specified by the culture producer. Just
before use, a container is thawed in warm (45°C), potable water and added to the raw material. The
directions for use of these cultures are supplied by the culture producers. 3
To produce freeze-dried concentrates, the liquid cell concentrates are shell frozen (in thin layers or small droplets) in either dry ice–acetone or in liquid nitrogen and then dried under vacuum.
The dried materials are packed in plastic bags under vacuum and stored at –20°C or at refrigerated
temperatures. They are transported to processors for quick delivery, either at ambient temperature
or in boxes containing ice packs. A processor stores the dried cultures at –20°C or in a refrigerator and uses them within the specified time. Just before use, the dried culture is mixed with warm
water (preferably boiled and cooled or sterile) and added to the raw materials. 3
To reduce costs in starter culture preparation, other drying methods, such as spray drying,
vacuum drying, drum drying, fluidized bed drying, and air drying methods, are now considered. 2,4 During spray drying, particles are formed as they are dried. Liquid cultures mixed with
a carrier molecule, such as gelatin, skim milk, gum Arabic, or starch, are sprayed using a nozzle
or rotary atomizer to form droplets, which when come in contact with hot air in a chamber form
dry powder. The major consideration of such approach is the viability of the starter cultures for a
desirable period of time. 2
Starter-Culture Problems
Strain Antagonism
In mixed-strain cultures, in which a starter culture contains two or more strains, dominance of
one over the others under a given condition can change the culture profile quickly. Dominance
can result from an optimum growth environment or production of inhibitory metabolites (e.g.,
bacteriocins, acids, peroxides). This can affect product quality and increase starter failure through
phage attack. Culture producers test the compatibility between desirable strains and develop
mixed-strain cultures with only the compatible strains to avoid strain antagonism.
Loss of a Desired Trait
A strain carrying a plasmid-linked desired trait can lose the trait during storage, subculturing,
and under some growth conditions. Physical and chemical stress and long freezing times can also
result in loss of a trait. Genetic studies are being conducted to understand the mechanisms of the
stability of these traits. Several strains with better stability of some traits have been developed and
are being used. In the future, more such strains will be available.
Cell Death and Injury
The effective use of frozen and freeze-dried concentrated cultures, especially for direct use (such as
DVS cultures), depends on two important characteristics: (1) Cultures need to have large numbers
of viable cells, and (2) cells should have a short lag phase so that they can start multiplying very
suspending medium contains cryoprotective agents to reduce cell death and injury during freezing
and thawing. The cell suspensions in metal containers are frozen, either by using dry ice–acetone
(–78°C) or liquid nitrogen (–196°C), and stored at that temperature. They are transported to processors, preferably by air, in Styrofoam boxes with sufficient amounts of dry ice. A processor stores
the containers at –20°C or below and uses it within the time specified by the culture producer. Just
before use, a container is thawed in warm (45°C), potable water and added to the raw material. The
directions for use of these cultures are supplied by the culture producers. 3
To produce freeze-dried concentrates, the liquid cell concentrates are shell frozen (in thin layers or small droplets) in either dry ice–acetone or in liquid nitrogen and then dried under vacuum.
The dried materials are packed in plastic bags under vacuum and stored at –20°C or at refrigerated
temperatures. They are transported to processors for quick delivery, either at ambient temperature
or in boxes containing ice packs. A processor stores the dried cultures at –20°C or in a refrigerator and uses them within the specified time. Just before use, the dried culture is mixed with warm
water (preferably boiled and cooled or sterile) and added to the raw materials. 3
To reduce costs in starter culture preparation, other drying methods, such as spray drying,
vacuum drying, drum drying, fluidized bed drying, and air drying methods, are now considered. 2,4 During spray drying, particles are formed as they are dried. Liquid cultures mixed with
a carrier molecule, such as gelatin, skim milk, gum Arabic, or starch, are sprayed using a nozzle
or rotary atomizer to form droplets, which when come in contact with hot air in a chamber form
dry powder. The major consideration of such approach is the viability of the starter cultures for a
desirable period of time. 2
Starter-Culture Problems
Strain Antagonism
In mixed-strain cultures, in which a starter culture contains two or more strains, dominance of
one over the others under a given condition can change the culture profile quickly. Dominance
can result from an optimum growth environment or production of inhibitory metabolites (e.g.,
bacteriocins, acids, peroxides). This can affect product quality and increase starter failure through
phage attack. Culture producers test the compatibility between desirable strains and develop
mixed-strain cultures with only the compatible strains to avoid strain antagonism.
Loss of a Desired Trait
A strain carrying a plasmid-linked desired trait can lose the trait during storage, subculturing,
and under some growth conditions. Physical and chemical stress and long freezing times can also
result in loss of a trait. Genetic studies are being conducted to understand the mechanisms of the
stability of these traits. Several strains with better stability of some traits have been developed and
are being used. In the future, more such strains will be available.
Cell Death and Injury
The effective use of frozen and freeze-dried concentrated cultures, especially for direct use (such as
DVS cultures), depends on two important characteristics: (1) Cultures need to have large numbers
of viable cells, and (2) cells should have a short lag phase so that they can start multiplying very